Mitochondrial fission and fusion require recruitment, assembly and disassembly of large protein complexes, but very few contributing proteins and their functions are known. Members of the dynamin family are key players: Drp1 is a cytosolic dynamin-related protein that mediates fission, Mitofusins are mitochondrial outer membrane dynamin-related proteins that mediate outer membrane fusion and Opa1 is an intermembrane space dynamin- related protein that mediates inner membrane fusion. We focus on the mechanisms involved in these processes and the cellular defects that arise when they are disrupted. In Aim 1, we propose to investigate alternative pathways for mitochondrial fission in C. elegans. We discovered that mitochondria in gonads of C. elegans drp-1 null alleles have seemingly wildtype mitochondria, which suggests that fission can occur without Drp1. This novel pathway could be specific for germlines in higher eukaryotes. We will investigate the mechanisms of Drp1 independent fission using live cell imaging, genetics and biochemistry. In Aim 2, we propose to investigate Mff function in mammalian cells. Mff is a novel tail-anchored protein required for mitochondrial fission in mammalian cells. Mff may affect Drp1 targeting to mitochondria or it may affect some other aspect of the scission process. We will investigate possible interactions between Mff, Drp1 and other mitochondrial fission proteins. These experiments will help determine the function of Mff and its place in the mitochondrial fission process. In Aim 3, we propose to investigate inducible proteolysis of Opa1 in mammalian cells. Opa1 is subjected to complex proteolytic processing in mammals. Inducible proteolysis during apoptosis inactivates Opa1, which could facilitate the release of cytochrome c from mitochondria. New data from our lab suggests that inducible cleavage is mediated by a protease called Oma1. We will verify this and we will explore possible synergy with other mitochondrial proteases. These experiments will help elucidate a novel pathway for inducible proteolysis in mammals. In Aim 4, we propose to investigate the mechanisms of Opa1 pathogenesis. We discovered that C. elegans Opa1 mutants are highly sensitive to damage from Reactive Oxygen Species (ROS). ROS also provides a plausible mechanism for pathogenesis in patients with dominant optic atrophy (a degenerative eye disease caused by mutations in Opa1). We will test this and alternative hypotheses for pathogenesis (faulty segregation of mtDNA or a direct role in apoptosis). We will also determine whether components of the electron transport chain produce more ROS in C. elegans Opa1 mutants. These experiments will help determine the relevance of ROS for optic atrophy and begin to address the underlying mechanisms of pathogenesis. Together our experiments will broaden our knowledge of mitochondrial fission and fusion, and provide new avenues for treatments of mitochondrial diseases. PUBLIC HEALTH RELEVANCE: Mitochondria are the power plants in cells. These small compartments frequently divide and fuse with each other. Mitochondrial fission and fusion play critical roles in many diseases, ranging from cancer to peripheral neuropathies and blindness, but the mechanisms underlying these processes are poorly understood. We focus on finding and analyzing new proteins that contribute to these processes, thus laying a foundation for research applied to human disease.